Sour cherry topical biotherapeutic formulations, method of manufacture and method of treatment of human osteoarthritis symptoms

ABSTRACT

A biotherapeutic anti-inflammatory composition, its method of manufacture and method of treatment of various diseases, the composition having an efficacious amount of solid sour cherry seed extract and sour cherry seed oil sufficient to inhibit production of disease-associated inflammatory cytokines by CD3+ T lymphocytes in a vertebrate mammal.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/059,232, filed October 3, 2014, and is adivisional application of U.S. patent application No. 15/923,253,issuing as U.S. Pat. No. 10,576,119, on Mar. 3, 2020, which was adivisional application of pending U.S. patent application Ser. No.14/874,065, filed Oct. 2, 2015.

BACKGROUND OF THE INVENTION

This invention relates in a general sense to topical biotherapeuticformulations, methods of manufacture of such formulations, and methodsof treatment of human osteoarthritis and other inflammatory diseases. Ina more particular sense, the invention relates to such formulations andmethods that utilize compositions extracted from sour cherry seeds.

Osteoarthritis (OA) treatments presently rely on analgesics, whichmanage pain but fail to restore imbalances between catabolic andanabolic processes underlying OA pathogenesis. Previously developedbiotherapeutic drugs (also known as biologics or biopharmaceuticals),which alter the activity of catabolic agents such as nitric oxide andinflammatory cytokines and allow tissue regeneration, were evaluated forefficacy in OA treatment. These studies failed to demonstrate dramaticabatement of OA symptoms by such drugs.

Osteoarthritis (OA), a degenerative age-related disease that affects thejoints, is the most common human musculoskeletal disorder, and a leadingcause of disability in elderly populations worldwide. OA onset istypically triggered by sustained biomechanical trauma, resulting inchondrocyte-mediated cartilage destruction.

Oxidative stress, created by this degradative process, promotesemergence of senescent osteoarthritic osteoblasts, which in turn enhancedysregulation of pro-inflammatory signaling and apoptotic depletion offunctional joint cells, causing insufficient cartilage repair andaberrant remodeling of the extracellular matrix. Tissue damage isexacerbated by trauma-related dysregulation of normal maintenance ofhealthy joint homeostasis. This disruption promotes increasingly severeinflammation (synovitis), leading to adverse changes in joint fluidcomposition, breakdown of extracellular matrix material, and impairmentof normal tissue repair.

The pathomechanisms of OA are facilitated by progressively elevatedlevels of the inflammatory cytokines TNF-α, and the interleukins (IL)IL-1β, IL-6, and IL-8, produced primarily by macrophages and Tlymphocytes, systemically and in affected joint tissue. Signalingcascades downstream of these cytokines also increases expression ofnitric oxide (NO) by mesenchymal cells (Volpi and Maccari 2005).Collectively, each of these factors contribute to joint capsulethickening, along with loss of cartilage, chondrocyte apoptosis,progressive articular dysfunction, and extreme chronic pain.

Several well-known features of the OA disease process that present veryattractive therapeutic targets are illustrateded in FIG. 1. For example,the production of inflammatory cytokines by activated CD3+ T lymphocytesoffers an excellent “choke point” for intervention in OA pathogenesis.This is due to critical roles for these mediators in disease-associatedpain and articular tissue destruction. Many treatments interfere withinflammatory cytokines at the level of their interaction with theirnormal physiological receptors and block downstream signaling processes,including dysregulated inflammation. However, a class of agents known asbiotherapeutic drugs is distinguished by mechanisms that modulatecellular signaling pathways to interfere with disease progression,promoting activities that contribute to healthy homeostasis. Theseapproaches differ from use of analgesics and related drugs currentlyfavored in clinical practice, which may ameliorate pain and othersymptoms, but have a negligible effect on the fundamentalpathomechanisms of OA.

Previous clinical trials of inflammatory cytokine inhibitors reported byother investigators failed to produce dramatic improvement of OAprognoses Inhibitors of both TNF and IL-1β were constructed as fusionproducts of a synthetic genetic element containing a portion of the genefor cognate receptors of TNF or IL-1β, spliced to the Fc (constant)portion of the Immunoglobin Gl (IgGl) antibody. The resulting fusionprotein binds to each cytokine, competitively reducing theirphysiological availability, thereby inhibiting their pro-inflammatoryeffects. In OA, this includes destruction of joint tissue (FIG. 1).These agents, administered systemically or via intra-articular injectionto OA patients affected in both knees and hands, failed to haltstructural deterioration or severity of symptoms. Moreover, geneticallyengineered cytokine inhibitors are extremely costly. For instance,per-patient costs for a one-year regimen of Etanercept, a TNF-inhibitoryfusion protein with broad application in inflammatory disease, isapproximately $20,000. This class of drug and related products is alsoassociated with severe side effects, including cancer and, occasionally,fatal immune impairment.

The lack of effective biopharmaceutical strategies for OA management hasleft clinicians heavily dependent on corticosteroids and non-steroidalanti-inflammatories. Despite being highly effective in controllinginflammation, these drugs are often severely toxic, particularly overextended time periods. Conversely, biotherapeutic strategies avoid smallmolecule inhibitors of pro-inflammatory signaling cascades—which may becostly, toxic, and only marginally effective.

It is an object of this invention to provide a novel formulation orcomposition of matter comprising the combination of sour cherry seedextract (SCE) and sour cherry seed oil (SCO) as an inducer of hemeoxygenase-1 (HO-1), a major physiological protectant against oxidativestress, in order to significantly reduce joint pain and activation ofCD4+ T cells expressing inflammatory cytokines (p<0.05); tosignificantly decrease peripheral blood c-reactive protein (CRP); and tosignificantly increase leukocyte HO-1 (p<0.05). The composition inhibitsjoint-damaging inflammatory mediator production, thereby meeting themain criterion for classification as a biotherapeutic, i.e., an agentthat inhibits disease pathogenesis rather than merely managing pain orreducing symptoms. It is a further object to provide a method ofmanufacture whereby the composition is formulated as a topical cream,and a further object to provide a method of treatment by topicalapplication of the composition wherein systemic concentrations ofbioactive compounds are delivered transdermally to ameliorateinflammation. It is a further object to provide such a compositionproduced by the process set forth herein.

SUMMARY OF THE INVENTION

In one aspect or embodiment, the invention is a topical creamcomposition comprising a stable mixture of sour cherry seed extract(SCE) and sour cherry seed oil (SCO), an emulsion comprising the solid,flavonoid-rich fraction of sour cherry (Prunus cerasus) seed suspendedin the oil fraction of the sour cherry seed. The solid, flavonoid-richfraction (SCE) and the oil fraction (SCO) are obtainable as separatecomponents by methods known in the art. A novel method of manufacture isrequired to produce the topical composition as an even and phase-stablesuspension in oil of multiple compounds of differential solubility. Thiscomposition contains natural excipients, such as oleic acid, whichfacilitate transdermal migration of bioactive compounds, resulting insystemic concentrations of these compounds high enough to cause thedesired physiologic effects.

In another aspect of embodiment, the invention is a method ofsystemically controlling inflammatory signaling in vertebrate animals(mammals and humans) to levels that prevent or mitigate tissue damage inall disease states in which symptoms result from dysregulatedinflammation, using a topically-applied biotherapeutic composition ofsour cherry seed kernel emulsion comprising a mixture of theflavonoid-rich solid fraction of the seed (SCE) and the oil of the seed(SCO).

In another aspect or embodiment, the invention is a method, and thecomposition produced by this methis, in which a phase-stable emulsion ismanufactured that disperses the bioactive components of the solidfraction of sour cherry (Prunus cerasus) seed kernel (SCE) in an oilphase, which comprises sour cherry seed oil (SCO) and other selectedoils suitable for human cosmetic use. Briefly, a lipophilic componentand a separate hydrophilic component are prepared, melted separately andthen combined in the melted state to create a cream base. After thecream base has cooled, the SCE and SCO active ingredients are added andmixed in.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of major pathomechanisms contributing toOA-associated articular tissue damage in a human joint. Mechanicaltrauma and endogenous oxidative stressors increase synoviocyte, T cell,and macrophage expression of the inflammatory cytokines IL-1β, TNF-α,and IL-6, resulting in aberrant chondrocyte activity and damagedcartilage. Increased macrophage TGF-β expression stimulates bone tissueosteophytes, promoting joint inflammation. Pathologically elevatedmacrophage activity also increases neovascularization, increasingproduction of pro-matrix metaloproteinase (ProMMP), which activates itscollagenase function by autocatalytic cleavage, allowing it tocontribute to OA-associated collagen matrix destruction.

FIG. 2 is a graph showing the effect of topical application of the sourcherry composition on OA-associated pain.

FIG. 3 is a graph showing the effect of topical application of the sourcherry composition on lymphocyte associated HO-1 production in PBMC fromOA patients.

FIG. 4 is a graph showing the effect of topical application of the sourcherry composition on serum c-reactive protein (CRP) levels in OApatients.

FIG. 5a is a graph showing the effect of topical application of the sourcherry composition on CD 4+ T lymphocyte expression of IL-8, TNF-a, andIFN-Y in OA patients.

FIG. 5b is a graph showing the effect of topical application of the sourcherry composition on CD 4+ T lymphocyte expression of IL-1α, IL-1β, andIL-6 in OA patients.

DETAILED DESCRIPTION

In general, the composition of the invention is a topically applied,biotherapeutic blend of phytochemicals that interferes with OApathogenesis by reducing the number of T lymphocytes activated toproduce disease-associated inflammatory cytokines. The combination ofsour cherry seed extract (SCE) and sour cherry seed oil (SCO)systemically increases the activity of HO-1, a major naturally-occurringantioxidant defense enzyme, ameliorates OA symptoms, and inhibitsactivation of CD3+ T cells to express inflammatory cytokines. HO-1 doesnot directly inactivate reactive oxygen molecules. Rather, itscontribution to antioxidant defense occurs indirectly due to two majorheme degradation products of HO-1 activity: bilirubin and carbonmonoxide (CO). The enzyme metabolizes heme that accumulates in tissuesas a result red blood cell turnover to CO, free iron and bilverdin.Biliverdin is rapidly reacted to bilirubin by ubiquitously expressedbiliverdin reductase, and in nanomolar intracellular concentrations actsas a powerful scavenger of reactive oxygen molecules. CO exerts itscytoprotective effects through increases in the activity of guanylatecyclase to increase levels of the second messenger cyclicguanosine-3′5′-monophosphate (cGMP), which interact with multiplecellular targets to reduce oxidative stress. SCE/SCO-induced HO-1inhibits the representation of CD3+ TNF-α+ and CD3+ IL-8+ cells incultured leukocytes from human rheumatoid arthritis and diabetes type 2patients at significantly greater levels than in cells from healthycontrol subjects. These findings are highly encouraging, as bothrheumatoid arthritis and type 2 diabetes are chronic inflammatorydiseases with many features of pathogenesis—particularly inflammatorytissue destruction—in common with OA.

The terms “biotherapetic”, biologic” or “biopharmaceutical” define anagent that alters the fundamental underlying cause of a disease, ratherthan simply treating symptoms on a temporary basis. This definition isgiven to the terms as used herein.

The topically administered SCE/SCO composition described herein isconsidered a biotherapeutic because it directly inhibits the activity ofinflammatory cytokines, which are core contributors to the disease.Components of the SCE/SCO composition curtail cytokine expression by Tcells via increased tissue activity of HO-1. Studies suggest thatphytochemical inducers of this enzyme are potentially superior to fusionprotein biopharmaceuticals, which inhibit cytokine activity at the levelof interaction with their cognate receptors.

The invention relates to control of inflammation that has becomedysregulated in ways that result in tissue damage characteristic of awide variety of diseases. The invention is a topical preparation inemulsion form of phytochemicals which penetrate the skin and becomesystemically bioavailable in efficacious concentration to significantlyinhibit primary immune activation that is responsible for inflammatorydisease symptoms. Diseases which exhibit features that this inventionhas been shown to therapeutically affect, include, but are not limitedto the following representative disorders: Osteoarthritis, RheumatoidArthritis, Allergic Rhinitis, Multiple Sclerosis, Uveitis, AutoimmuneDiabetes (type I, IDDM), Type 2 Diabetes (metabolic syndrome), BronchialAsthma, Psoriasis Vulgaris, Eczema, Systemic Lupus Erythematosus (SLE),Alopecia Aereata, Hashimoto's Thyroiditis, IgA Nephropathy, Hydrops,Kell Erythroblastosis Fetalis and Pemphigus. 20

All diseases that include underlying pathomechanisms caused bydysregulated inflammatory processes exhibit common features that may beexploited in strategies for prevention and therapy. It has been shownthat the SCE/SCO composition described herein affects activation of CD3+T cells in ways that significantly ameliorated joint pain anddisease-associated biomarkers in human OA patients. Osteoarthritis wasselected as a model disease for demonstration of this invention since ithas core pathomechanisms in common with a wide range of disorders, butthe invention is applicable to other inflammation-inducing diseases aswell.

OA is a degenerative age-related disease that affects the joints, is themost common human musculoskeletal disorder, and is a leading cause ofdisability in elderly populations worldwide. OA onset is typicallytriggered by sustained biomechanical trauma, resulting inchondrocyte-mediated cartilage destruction. Oxidative stress, created bythis degradative process, promotes emergence of senescent osteoarthriticosteoblasts, which in turn enhance dysregulation of pro-inflammatorysignaling and apoptotic depletion of functional joint cells, causinginsufficient cartilage repair and aberrant remodeling of theextracellular matrix. Tissue damage is exacerbated by trauma-relateddysregulation of normal maintenance of healthy joint homeostasis. Thisdisruption promotes increasingly severe inflammation (synovitis),leading to adverse changes in joint fluid composition, breakdown ofextracellular matrix material, and impairment of normal tissue repair.The pathomechanisms of OA are facilitated by progressively elevatedlevels of the inflammatory cytokines TNF-α, and the interleukins (IL)IL-1β, IL-6, and IL-8, produced primarily by macrophages and Tlymphocytes, systemically and in affected joint tissue. Signalingcascades downstream of these cytokines also increases expression ofnitric oxide (NO) by mesenchymal cells. Collectively, each of thesefactors contribute to joint capsule thickening, along with loss ofcartilage, chondrocyte apoptosis, progressive articular dysfunction, andextreme chronic pain.

In one aspect or embodiment, the invention is a topical creamcomposition comprising sour cherry seed extract (SCE) and sour cherryseed oil (SCO), presented as an emulsion comprising the solid,flavonoid-rich fraction of sour cherry (Prunus cerasus) seed suspendedin the oil fraction of the sour cherry seed. The solid, flavonoid-richfraction (SCE) and the oil fraction (SCO) are obtainable as separatecomponents for subsequent mixing by methods known in the art. Inparticular, methodology for obtaining SCE and SCO is set forth in Bak Iet al. (2010) “Isolation and analysis of bioactive constituents fo sourcherry (Prunus cerasus) seed kernel; an emerging functional food.” J.Med. Food 13:905-910, the disclosure of which is incorporated herein byreference.

A novel method of manufacture is required to produce the SCE/SCOcomposition as an even and phase-stable suspension in oil of multiplecompounds of differential solubility, as prior efforts have resulted inundesirable precipitation. This composition contains natural excipients,such as oleic acid, which facilitate transdermal migration of bioactivecompounds, resulting in systemic concentrations of these compounds highenough to cause the desired physiologic effects.

In another aspect of embodiment, the invention is a method ofsystemically controlling inflammatory signaling in vertebrate animals tolevels that prevent or mitigate tissue damage in all disease states inwhich symptoms result from dysregulated inflammation, using atopically-applied biotherapeutic composition comprising a mixture of thesolid (i.e., powder form) flavonoid-rich solid fraction of the seed(SCE), the oil fraction of the seed (SCO) and other selected compounds.

In another aspect or embodiment, the invention is a method or processand a composition produced by this method or process in which aphase-stable emulsion is manufactured that disperses the bioactivecomponents SCE and SCO of the sour cherry (Prunus cerasus) seed. Theprocess for extracting the kernel contents of sour cherry seeds,separation of the oil fractions and preparation of the powdered extractused to prepare the topical product is known in the art.

The method of manufacture comprises the steps of:

(a) creating a lipophilic component comprising a mixture of variousingredients;

(b) creating a hydrophilic component comprising a mixture of variousingredients;

(c) heating said lipophilic component to a sufficient temperature tomelt or liquefy said lipophilic component and separately heating saidhydrophilic component to a sufficient temperature to melt or liquefysaid hydrophilic component;

(d) combining and mixing both components while at the same temperatureand with all the components in the melted or liquified state andallowing the mixed components to cool to ambient temperature to form acream base;

(e) adding the sour cherry seed oil (SCO) into the cream base andmixing;

(f) adding sour cherry seed extract SCE) in powdered fonn into the creambase and mixing.

In a preferred embodiment, the two components are each heated separatelyin a water bath at a temperature of approximately 50-60° C. The SCE/SCOtopical composition should be stored in a dark, cool place atapproximately 2-8° C. Preferably the solid sour cherry seed extract(SCE) and the solid cherry seed oil (SCO) are each present in theSCE/SCO composition at approximately 3-5 wt %.

Prior attempts at mixing the solid, flavonoid-rich fraction (SCE) andthe oil fraction (SCO) have not been successful in producing a highlystable mixture. The methodology set forth above, however, produces ahighly stable emulsion composition with evenly blended sour cherry seedlipid soluble components and water soluble components. The SCE/SCOcomposition so produced is a highly efficient and effectivebiotherapeutic, anti-inflammatory, topical cream base composition.

A representative and efficacious biotherapeutic SCE/SCO composition maycomprise the following ingredients, presented by weight percent and withillustrative weights for production of a 5000 gram (g) batch of thecomposition, the weight percentages and weights being approximate:

Wt Percent Weight 5 wt % 250 g lanolin (a.k.a wool fat) 5 wt % 250 gcetylic and stearylic alcohol 10 wt % 500 g stearin 5 wt % 250 gisopropyl myristate 0.8 wt %  40 g vitamin C 2 wt % 100 g sodium laurylsulphate 5 wt % 250 g glycerin 5 wt % 250 g propylene glycol 52.2 wt %2610 g  distilled water 5 wt % 250 g sour cherry seed oil (SCO) 5 wt %250 g powdered sour cherry seed extract (SCE)

The lipophilic component in this example consists of the mixture oflanolin, cetylic and stearylic alcohol, stearin and isopropyl myristate.Based on the weight percentages above, the lipophilic componentcomprises approximately 20 wt % lanolin, approximately 20 wt % cetylicand stearylic acid, approximately 40 wt % stearin and approximately 20wt % isopropyl myristate. The hydrophilic component in this exampleconsists of the mixture of vitamin C, sodium lauryl sulphate, glycerin,propylene glycol and distilled water. The hydrophilic componentcomprises approximately 1.2 wt % vitamin C, approximately 3.1 wt %sodium lauryl sulphate, approximately 7.7 wt % glycerin, approximately7.7 wt % propylene glycol, and approximately 80.3 wt % water.

The SCE/SCO composition as described has been found to provide thefollowing when topically applied to vertebrate mammals, includinghumans:

(a) Significant suppression (p<0.05) of osteoarthritis-associated jointpain.

(b) Significant inhibition (p<0.05) of the inflammation-associated serumbiomarker of inflammation, c-reactive protein (CRP).

(c) Significant increased (p<0.05) expression of heme oxygenase-1 (HO-1)by peripheral blood leukocytes to therapeutic levels.

(d) Significant inhibition (p<0.05) of production of disease-associatedinflammatory cytokines by CD3+ T lymphocytes at levels capable ofameliorating symptoms of osteoarthritis.

The method of treatment comprises the steps of applying the SCE/SCOcomposition as described and manufactured above topically to the skin ofa vertebrate mammal in repeated applications such that the SCE and SCOingredients migrate into and through the skin to an area ofinflammation.

The SCE/SCO composition addresses the type of inflammation associatedwith Osteoarthritis, Rheumatoid Arthritis, Allergic Rhinitis, MultipleSclerosis, Uveitis, Autoimmune Diabetes (type I, IDDM), Type 2 Diabetes(metabolic syndrome), Bronchial Asthma, Psoriasis Vulgaris, Eczema,Systemic Lupus Erythematosus (SLE), Alopecia Aereata, Hashimoto'sThyroiditis, IgA Nephropathy, Hydrops, Kell Erythroblastosis Fetalis andPemphigus.

The beneficial and efficacious results of the topical application of theSCE/SCO composition as described herein has been demonstrated in arepresentative clinical trial directed to the treatment ofosteoarthritis (OA):

Study Participants

Participants included 30 patients aged 40 years or older, diagnosed withinflammatory OA of the knee, according to criteria for OA set by theAmerican College of Rheumatology. Patients selected for participation inthe study were under treatment regimens that included non-steroidalanti-inflammatory agents. Persons with medical conditions which mightaffect outcome measures independent from OA pathogenesis or routinetreatment for the condition were excluded. The present study met KuwaitUniversity's human subject protection criteria and was approved by theUniversity's Institutional Review Board (IRB)/ethics committee. Informedconsent agreements were obtained from all participating subjects.

Each subject was treated topically with 5 ml of the SCE/SCO compositiontwice daily for 2 months. Treatments were conducted by applying 2.5 mlof the cream to both knees of each participant, followed by dispersalacross the kneecap and surrounding skin in a circular motion, continuinguntil the full 5 ml had been absorbed. Patients in the placebo groupwere administered 5 ml of a variety of the skin cream created from theseed oil without the flavonoid fraction. The SCE/SCO composition wasprepared from seed kernels as previously described.

Treatment Groups and Outcomes Assessed

The participants were randomly assigned into one of 2 treatment groupsdefined as follows: SCE GROUP: Twenty subjects, administered the SCE/SCOcomposition twice daily for 2 months; CONTROL (PLACEBO) GROUP: Tensubjects, administered a sour cherry oil-based vehicle without the solidflavonoid fraction (SCE) twice daily for 2 months.

Patients were assessed at baseline and at week 8 of treatment for: (i)Index knee pain using the WOMAC pain subscale (Bellamy et al. 1988);(ii) Serum HO-1; (iii) Serum c-reactive protein (CRP); (iv) Activationof T lymphocytes to express the inflammatory cytokines IL-8, TNF-α,IFN-γ, IL-1α, IL-1β and IL-6. Blood collection and analysis wasconducted according to previously published methods used by thislaboratory. Statistical analyses of data were performed using WindowsNorusis/SPSS version 17. A p-value of <0.05 was considered statisticallysignificant.

Clinical and Laboratory Outcomes

Prior to study enrollment, each patient was screened for compliance withinclusion/exclusion criteria. Parameters evaluated included medicalhistory, current health status (by physical examination), laboratory andradiographic evaluations and a review of current medications. Themethodology for measurement of each of these outcomes is summarizedbelow:

(a) Pain assessment: Self-assessed pain in the index knee was measuredusing the 100-mm visual analogue scale (VAS) on the WOMAC pain subscale.

(b) Phlebotomy and extraction of peripheral blood mononuclear cells(PBMC): 10-ml samples of sodium heparin-anticoagulated peripheral venousblood were collected from study participants in Vacutainer collectiontubes (Becton Dickinson Biosciences Inc., Rutherford, N.J., USA) anddiluted 1:1 in sterile phosphate-buffered saline (PBS) followedimmediately by isolation of PBMC using density centrifugation onFicoll-Hypaque gradients (Pharmacia, Uppsala, Sweden); and a Centra-CL-2centrifuge (MidAtlantic Diagnostics, Inc., Mount Laurel, N.J. 08054USA).

(c) Cell culture: PBMC were separated by Ficoll-paque (Pharmacia,Uppsala, Sweden) density gradient centrifugation. The cells were washedand suspended in RPMI 1640 medium (Gibco BRL, Gaithersburg, Md., USA) atdensity of 1×10(6) cells/ml. 200 μl cultures in 96-well plates wereincubated under humidified conditions for 6 hours at 37° C. in anatmosphere of 5% CO2. PBMC were stimulated in the presence of 50 ng/mlof phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, Mo.), 1 ng/mlof ionomycin (Sigma) and 2 mM monensin (Sigma). Here, monensin is addedto cells as a glycoprotein export inhibitor, which allows intracellularaccumulation of each target cytokine, thus enhancing its signal duringflow cytometric analysis.

(d) Flow Cytometric Analysis for Inflammatory Cytokines: Expression ofIL-8, IFNγ, IL1-α, IL1-β and IL6 in CD3+ CD4+ lymphocytes in freshlycollected peripheral blood or for each cell culture stimulationcondition was analyzed. Briefly, cells harvested from each culture werefirst incubated for 15 minutes at room temperature withfluorescein-isothiocyanate (FITC) anti-human CD3+ (Dakopatts, A/S,Glostrup, Denmark), then fixed and permeabilized using the Fix and Permcell permeabilization kit (Life Technologies Inc., Eugene Oreg.,U.S.A.). Intracellular labeling of permeablized cells for inflammatorycytokines was accomplished by 30-minute incubations at room temperature,with phycoerythrin (RD1)-conjugated monoclonal antibodies to human IL-8IFNγ, IL1-α, IL1-β and IL6 (BD PharMingen, Heidelberg, Germany). PBMCswere then washed and evaluated by 2-color flow cytometry for expressionof each selected cytokine using the FC-500 flow cytometer (BeckmanCoulter Corporation, Hialeah, Flor., U.S.A.). Isotypic controls for theantibody used to detect cytokine expression were established for eachcell preparation. Positive analysis regions for cells expressingselected CD (cluster of differentiation) immunophenotypic markers andcytokines were set against controls; and specific binding offluorophore-conjugated antibodies was analyzed according to standardmethods recommended by the manufacturer. Lymphocyte subpopulations wereidentified by position on forward and side scatter plots. Staining ofcell surface and internal antigens of interest in preparation for flowcytometry was conducted according to the manufacturer's protocol(InVitrogen Molecular Probes manual: DETECTION OF INTRACELLULAR ANTIGENSBY FLOW CYTOMETRY (Rev 03/10) DCC-10-0815 (PN 624923BD). Issue A InitialIssue, 8/03 Rev Issue BD 10/11, Cytomics FC 500 CXP Software IFU Manual,Running Samples Sec 4.1 & Creating Protocols Section 1-19).

(e) ELISA analysis for HO-1 expression. Measurement of lymphocyteexpression of HO-1 was made using the StressXpress™ Human HO-1 ELISA Kit(Enzo Life Sciences International, Inc., Plymouth Meeting, Pa., USA).Briefly, lysates made from cells were incubated in 96-well microtiterplates coated with anti-human HO-1 antibody, followed by treatment withsecondary/detect antibody and related reagents provided with kits.Cell-associated HO-1 expression was evaluated during the absorbance ofthe developed kit reagents at 450 nm in a Biotek ELX 808 MicroplateReader. Results are reported as mean values in ng/ml of HO-1 inlymphocyte lysates of each patient group +standard error of the mean(SEM).

ELISA analysis for c-reactive protein (CRP) levels. Measurement of CRPwas made by using Active US® CRP ELISA Kit (Diagnostic SystemLaboratories, Inc. Webster, Tex., USA). Briefly, peripheral blood wascollected in non-anticoagulated vacutainer tubes (Becton Dickinson Inc.)and allowed to stand at room temperature for 2 hours to form clots, fromwhich serum was extracted with sterile applicators. Serum samples weresubsequently incubated in 96-well microtiter plates coated with anti-CRPantibody, followed by treatment with horseradish peroxidase(HRP)-conjugated anti-CRP. CRP concentration in each sample which wasproportional to HRP-mediated conversion of a colorimetric substrate wasestimated by monitoring of dual wavelength absorbance at 450 and 620 nmusing a Synergy HT Multi-Mode Micro plate Reader.

(g) Statistical analysis. Wilcoxon signed ranks test was used to comparevariables in each group before and after treatment. Correlations betweenvariables within each group were performed using Spearman rankcorrelation test. The analyses were performed using the SPSS for Windowsstatistical package version 17 (Norusis/SPSS, Inc.). A P-value of <0.05was considered statistically significant.

Results Clinical Outcomes

The effect of topical SCE/SCO treatment on joint pain, self-assessed byOA patients in a selected index knee is shown in FIG. 2. When comparedto pain scores measured before treatment (baseline values),SCE/SCO-treated patients reported significantly decreased pain following2 months of daily application of the product (p<0.001). In contrast,comparison of pain scores reported by patients in the placebo group atbaseline, with scores following 2 months of treatment with the placeboskin cream, revealed only non-significant differences (p=0.139).

FIG. 3 shows expression of leukocyte-associated HO-1 in test and controlparticipants. Measurement of HO-1 content of PBMC lysates taken fromboth groups reveals significant increases lymphocyte content of theenzyme in cells from SCE/SCO-treated OA patients evaluated by ELISAfollowing 2 months of treatment versus baseline levels (p<0.05). Anon-significant difference in baseline versus post-treatment PBMC HO-1levels was revealed for the placebo patients (p=0.220).

Evaluation of c-reactive protein (CRP) in peripheral blood serum of OApatients after 2 months of topical SCE treatment revealed significantlylower levels of this inflammatory biomarker than in samples taken in thesame group at baseline (p<0.005, FIG. 4). Comparison of serum CRP inplacebo-treated subjects showed only non-significant differences betweenbaseline and post-treatment values (p=0.957).

The effect of SCE/SCO treatment on activation of peripheral bloodleukocytes is shown in FIG. 5. Here, the effect of treatments on CD4+ Tcells is shown, since these cells are a major source of inflammatory thecytokines contributing to OA pathogenesis. It is neverthelessanticipated that SCE/SCO also alters pro-inflammatory signaling bymacrophages and other tissue. Ongoing studies are evaluating the scopeof SCE/SCO-mediated effects in other cell types. Relative to baselinemeasurements, PBMC from SCE/SCO-treated OA patients cultured with PMA/I,contained significantly lower post-treatment representation of CD4+IL-8+(p<0.001), CD4+ TNF-α+ (p<0.005) and CD4+ IFN-γ+(p<0.005) (5A),CD4+IL-1α+ (p<0.005) and CD4+IL-1β+ (p<0.005), but not CD4+IL-6(p=0.494) (5B). In these experiments, comparison of cells from blood ofOA patients taken at baseline, versus placebo-treated patients shownon-significant differences in representation of CD4+ T cells activatedto express IL-8 (p=0.730), TNF-α (p=0.165) and IFN-γ (p=0.160) (5A),IL-1α (p=0.620), (p=0.406) and IL-6 (p=0.240) (5B).

Implication of Preliminary Human Trials

The representative investigation described above was a double-blindclinical trial, evaluating a hypothesis that the SCE/SCO composition asset forth, administered topically, reduces joint pain and systemicallyinhibits expression of inflammatory cytokines that promote OApathogenesis; along with reduction in c-reactive protein (CRP), andnumbers of T cells activated to express IL-8, TNF-α, IFN-γ, IL-1α, andIL-1β. These results were obtained by persons administered extracts ofthe sour cherry seed kernel comprising both solid sour cherry seedextract (SCE) and solid cherry seed oil (SCO), but not the fruit itself.

Based on previous investigations and the significantly increasedlymphocyte HO-1 expression noted in SCE-treated patients relative tobaseline and placebo (FIG. 3) the therapeutic mechanisms likely includeHO-1-mediated quenching of proinflammatory reactive oxygen species. Inthis study, we observed that topical treatment of OA patients with aSCE/SCO preparation of sour cherry flavonoids that include HO-1 inducerswhich therapeutically diminish oxidative stress burden on tissuesignificantly abated major OA symptoms. These effects occurred viainhibition of CD3+ T cell expression of inflammatory cytokines, thusdemonstrating the capacity of this plant material to ameliorate OAseverity by altering a major underlying contributor to diseaseprogression.

Relevance of HO-1 Increases in SCE/SCO-Treated Subjects: In therepresentative human study described above, the significantly increasedexpression of HO-1 by PBMC, isolated from the blood of SCE-treatedsubjects (p<0.05) shown in FIG. 3, is consistent with the expectedeffect of transdermal delivery of HO-1-inducers at levels sufficient tomediate systemic up-regulation of the enzyme. Indeed, increased activityof HO-1 in immune cells (and possibly other tissue) may account for thesignificant SCE treatment-associated reduction in serum CRP shown inFIG. 4 (p<0.05), as CRP level is a well-known correlate of the severityof inflammatory diseases, including OA. These observations furtherunderscore the role of disease- or trauma-related HO-1 expression as ageneral adaptive response to dysregulated inflammation with therapeuticpotential in many clinical venues. It is therefore tempting to speculatethat SCE/SCO-induced increases in HO-1 expression is the criticalmediator of significant treatment-related decreases in therepresentation of CD3+ subpopulations activated to express theinflammatory cytokines IL-8, TNF-α, IFN-γ, IL-1α, IL-1β, and IL-6(p<0.05), shown in FIGS. 55a and 5 b.

It is understood that equivalents and substitutions for certain elementsand steps set forth above may be obvious to one of ordinary skill in theart, and therefore the true scope and definition of the invention is tobe as set forth in the following claims. The examples and any preferredembodiments as set forth above are meant to be non-limiting as to thescope of the invention.

We claim:
 1. A biotherapeutic anti-inflammatory composition produced bya process comprising the steps of: (a) providing a lipophilic componentcomprising at least some ingredients not present naturally in sourcherry seeds; (b) providing a hydrophilic component comprising at leastsome ingredients not present naturally in sour cherry seeds; (c)providing solid flavonoid-rich sour cherry seed extract in powderedform; (d) providing sour cheery seed oil; (e) heating said lipophiliccomponent to a sufficient temperature to melt or liquefy said lipophiliccomponent and separately heating said hydrophilic component to asufficient temperature to melt or liquefy said hydrophilic component;(f) mixing said melted or liquefied lipophilic component and said meltedor liquefied hydrophilic component; (g) allowing said mixed heatedlipophilic and hydrophilic components to cool to form a cream base; and(h) mixing said sour cheery seed oil and said solid flavonoid-rich sourcherry seed extract in powdered form into said cream base.
 2. Thecomnposition of claim 1, wherein said step of mixing said sour cherryseed oil and said solid flavonoid-rich sour cherry seed extract inpowdered form into said cream base comprises mixing an amount of saidsour cherry seed oil and an amount of said solid flavonoid-rich sourcherry seed extract in powdered form such that said sour cherry seed oilis approximately 3-5 wt % of said composition and said solidflavonoid-rich sour cherry seed extract in powdered form isapproximately 3-5 wt % of said composition.
 3. The composition of claim1, wherein said step of providing a lipophilic component comprises thestep of mixing lanolin, cetylic and stearylic alcohol, stearin andisopropyl myristate; and wherein said step of providing a hydrophobiccomponent comprises the step of mixing vitamin C, sodium laurylsulphate, glycerin, propylene glycol and water.
 4. The composition ofclaim 3, wherein said step of providing a lipophilic component comprisesthe step of mixing approximately 20 wt % lanolin, approximately 20 wt %cetylic and stearylic acid, approximately 40 wt % stearin andapproximately 20 wt % isopropyl myristate to form said lipophiliccomponent; and wherein said step of providing a hydrophilic componentcomprises the step of mixing approximately 1.2 wt % vitamin C,approximately 3.1 wt % sodium lauryl sulphate, approximately 7.7 wt %glycerin, approximately 7.7 wt % propylene glycol, and approximately80.3 wt % water to form said hydrophilic component.
 5. The compositionof claim 3, wherein said steps of providing a lipophilic component,providing a hydrophobic component, providing solid flavonoid-rich sourcherry seed extract in powdered foiiii, and providing sour cheery seedoil comprise providing approximately 5 wt % lanolin, approximately 5 wt% cetylic and stearylic alcohol, approximately 10 wt % stearin,approximately 5 wt % isopropyl myristate, approximately 0.8 wt % vitaminC, approximately 2 wt % sodium lauryl sulphate, approximately 5 wt %glycerin, approximately 5 wt % propylene glycol, approximately 52.2 wt %water, approximately 5 wt % solid flavonoid-rich sour cherry seedextract and approximately 5 wt % sour cherry seed oil relative to saidcomposition.
 6. The composition of claim 1, wherein said step of heatingsaid lipophilic component and separately heating said hydrophiliccomponent comprises heating each in a water bath at a temperature ofapproximately 50-60° C.
 7. A biotherapeutic anti-inflammatory topicalcomposition in the foini of a phase-stable emulsion, said compositioncomprising a mixture of solid flavonoid-rich sour cherry seed extract inpowdered form previously separated from sour cherry seeds and sourcherry seed oil previously separated from sour cherry seeds, whereinsaid solid flavonoid-rich sour cherry seed extract in powdered form andsaid sour cherry seed oil are present in amounts producing ananti-inflammatory effect, said composition altering the underlyingpathomechanisms caused by dysregulated inflammatory processes byinhibiting production of disease-associated inflammatory cytokines byCD3+ T lymphocytes, inhibiting the inflammation-associated serumbiomarker c-reactive protein, and increasing expression of hemeoxygenase-1 by peripheral blood leukocytes, when topically applied,produced by a process comprising the steps of: (a) providing alipophilic component comprising at least some ingredients not presentnaturally in sour cherry seeds; (b) providing a hydrophilic componentcomprising at least some ingredients not present naturally in sourcherry seeds; (c) providing solid flavonoid-rich sour cherry seedextract in powdered form; (d) providing sour cheery seed oil; (e)heating said lipophilic component to a sufficient temperature to melt orliquefy said lipophilic component and separately heating saidhydrophilic component to a sufficient temperature to melt or liquefysaid hydrophilic component; (f) mixing said melted or liquefiedlipophilic component and said melted or liquefied hydrophilic component;(g) allowing said mixed heated lipophilic and hydrophilic components tocool to form a cream base; and (h) mixing said sour cheery seed oil andsaid solid flavonoid-rich sour cherry seed extract in powdered form intosaid cream base.
 8. The composition of claim 7, wherein said step ofmixing said sour cherry seed oil and said solid flavonoid-rich sourcherry seed extract in powdered form into said cream base comprisesmixing an amount of said sour cherry seed oil and an amount of saidsolid flavonoid-rich sour cherry seed extract in powdered form such thatsaid sour cherry seed oil is approximately 3-5 wt % of said compositionand said solid flavonoid-rich sour cherry seed extract in powdered formis approximately 3-5 wt % of said composition.
 9. The composition ofclaim 7, wherein said step of providing a lipophilic component comprisesthe step of mixing lanolin, cetylic and stearylic alcohol, stearin andisopropyl myristate; and wherein said step of providing a hydrophobiccomponent comprises the step of mixing vitamin C, sodium laurylsulphate, glycerin, propylene glycol and water.
 10. The composition ofclaim 9, wherein said step of providing a lipophilic component comprisesthe step of mixing approximately 20 wt % lanolin, approximately 20 wt %cetylic and stearylic acid, approximately 40 wt % stearin andapproximately 20 wt % isopropyl myristate to form said lipophiliccomponent; and wherein said step of providing a hydrophilic componentcomprises the step of mixing approximately 1.2 wt % vitamin C,approximately 3.1 wt % sodium lauryl sulphate, approximately 7.7 wt %glycerin, approximately 7.7 wt % propylene glycol, and approximately80.3 wt % water to form said hydrophilic component.
 11. The compositionof claim 9, wherein said steps of providing a lipophilic component,providing a hydrophobic component, providing solid flavonoid-rich sourcherry seed extract in powdered form, and providing sour cheery seed oilcomprise providing approximately 5 wt % lanolin, approximately 5 wt %cetylic and stearylic alcohol, approximately 10 wt % stearin,approximately 5 wt % isopropyl myristate, approximately 0.8 wt % vitaminC, approximately 2 wt % sodium lauryl sulphate, approximately 5 wt %glycerin, approximately 5 wt % propylene glycol, approximately 52.2 wt %water, approximately 5 wt % solid flavonoid-rich sour cherry seedextract and approximately 5 wt % sour cherry seed oil relative to saidcomposition.
 12. The composition of claim 1, wherein said step ofheating said lipophilic component and separately heating saidhydrophilic component comprises heating each in a water bath at atemperature of approximately 50-60° C.